Antibacterial polypeptide AuGF and application thereof in preparation of medicine for treating and / or preventing bacterial infection

Abstract

The invention discloses an antibacterial polypeptide AuGF and application of the antibacterial polypeptide AuGF in preparation of a medicine for treating and / or preventing bacterial infection. The amino acid sequence of the polypeptide is as follows: SEQ ID NO.1: GLFIKIIKIKIAKSF; the antibacterial polypeptide AuGF disclosed by the invention has broad-spectrum antibacterial activity, can effectively solve the problem of bacterial drug resistance, and has a remarkable bacteriostatic effect on various bacteria including staphylococcus aureus, streptococcus mutans, actinobacillus, methicillin-resistant staphylococcus aureus and the like; the compound provided by the invention has the advantages of simple preparation process and low cost, can play a significant role at low concentration, has low minimum inhibitory concentration on various pathogenic bacteria, has strong bacteriostatic ability, and is beneficial to reduction of side effects and improvement of drug safety; the antibacterial polypeptide AuGF can also be used as a bacteriostatic additive to be applied to articles of daily use, provides an effective bacteriostatic means for daily cleaning and skin care, and has a wide market application prospect.

Description

Technical Field

[0001] The present invention relates to an antibacterial polypeptide AuGF, and also relates to the application of the above antibacterial polypeptide AuGF in the preparation of a drug for treating and / or preventing bacterial infections. Background Art

[0002] The widespread use and abuse of antibiotics have led to the rapid emergence of drug-resistant bacteria, which has become one of the major challenges facing global public health. With the continuous increase of drug-resistant strains, traditional antibiotic treatment has become increasingly difficult, which urgently requires us to search for new antibacterial strategies and methods. Antimicrobial peptides are part of the body's innate immune system. They are a class of small proteins with broad antibacterial activity. The antibacterial mechanism of antimicrobial peptides is different from that of traditional antibiotics. They usually kill bacteria through physical means rather than inhibiting specific metabolic pathways, which makes it difficult for bacteria to develop resistance to them. At the same time, the diversity and plasticity of antimicrobial peptides provide a broad space for the design of new antibacterial drugs.

[0003] Antimicrobial peptides are a class of amphiphilic polypeptides with cationic properties. They bind to the negatively charged cell membrane of bacteria through electrostatic interactions and aggregate on the membrane surface. After reaching the critical concentration, antimicrobial peptides can disrupt the integrity of the cell membrane, form transmembrane pores, and cause the leakage of bacterial contents, thereby inhibiting bacterial growth or directly causing bacterial death. In addition, antimicrobial peptides can also interfere with the physiological activities of bacteria through non-membrane-soluble mechanisms, such as interacting with DNA, RNA, or proteins inside bacteria. These polypeptides have a small molecular weight, a simple structure, are easy to synthesize, and usually do not cause an immune response. Therefore, they have broad antibacterial activity and a low risk of drug resistance, showing potential in the development of new antibacterial drugs. However, there are still the following key defects in their practical applications:

[0004] 1. Insufficient antibacterial activity: The literature "Evaluation of LL-37 antimicrobial peptide derivatives alone and in combination with vancomycin against S. aureus" (2018) tested the minimum inhibitory concentration (MIC) of the classic antimicrobial peptide LL-37 against MRSA (methicillin-resistant Staphylococcus aureus) to be as high as 256 μg / mL, which is difficult to meet the clinical low-dose requirements; the literature "Harnessing bacterial antimicrobial peptides: a comprehensive review on properties, mechanisms, applications, and challenges in combating antimicrobial resistance" (2024) pointed out that the MIC of most natural antimicrobial peptides against Gram-negative bacteria (such as Pseudomonas aeruginosa) exceeds 20 μmol / L, and the broad-spectrum property is limited.

[0005] 2. High synthesis cost and low efficiency: The long-chain cationic peptides (such as 30 - 40 amino acids) described in Patent CN 117603314 have a low solid-phase synthesis yield due to their complex sequences, and the purification cost is extremely high; the literature "Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo" (2017) shows that it is difficult to industrialize the production of antimicrobial peptides containing rare amino acids (such as D-configuration), and it is difficult to apply them on a large scale.

[0006] 3. Slow antibacterial effect: The Ib-M antimicrobial peptide reported in the literature "Antimicrobial activity of Ib-M peptides against Escherichia coli O157:H7" (2020) could not completely sterilize (99.9%) even after 4 hours at the MIC concentration.

[0007] In summary, the deficiencies of existing antimicrobial peptides in terms of activity, cost, and bactericidal speed restrict their clinical translation and daily life applications to a certain extent. Summary of the Invention

[0008] Object of the Invention: The object of the present invention is to provide an antibacterial polypeptide AuGF with a short chain, high antibacterial activity, and broad-spectrum antibacterial activity. The present invention also provides the use of the above antibacterial polypeptide AuGF in the preparation of drugs for treating and / or preventing bacterial infections, providing new ideas for the research and development of clinical anti-infective drugs, and being widely used as a bacteriostatic additive in daily necessities.

[0009] Technical Solution: The present invention discloses an antibacterial polypeptide AuGF. The amino acid sequence of the polypeptide is SEQ ID NO.1: glycine (G)-leucine (L)-phenylalanine (F)-isoleucine (I)-lysine (K)-isoleucine (I)-isoleucine (I)-lysine (K)-isoleucine (I)-lysine (K)-isoleucine (I)-alanine (A)-lysine (K)-serine (S)-phenylalanine (F) (abbreviation: GLFIKIIKIKIAKSF). The antibacterial polypeptide AuGF is an amphiphilic cationic antibacterial peptide, which binds to negatively charged phospholipid molecules on the bacterial biofilm through its positive charge and interacts with the bacterial cell membrane, thereby destroying the cell membrane structure of bacteria and achieving the effect of inhibiting bacterial growth. In addition, the antibacterial polypeptide shows low cytotoxicity and can be used in the preparation of drugs for treating and / or preventing bacterial infections.

[0010] The above antibacterial polypeptide AuGF or its pharmaceutically acceptable salt can be used in the preparation of drugs for treating and / or preventing bacterial infections.

[0011] Among them, the bacteria are Gram-positive bacteria and / or Gram-negative bacteria.

[0012] Among them, the bacteria include one or more of Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, or methicillin-resistant Staphylococcus aureus.

[0013] Among them, the minimum inhibitory concentration of the antibacterial polypeptide AuGF against Staphylococcus aureus is 1 μmol / L.

[0014] Among them, the minimum inhibitory concentration of the antibacterial polypeptide AuGF against Streptococcus mutans or Actinobacillus actinomycetemcomitans is 5 μmol / L.

[0015] Among them, the minimum inhibitory concentration of the antibacterial polypeptide AuGF against methicillin-resistant Staphylococcus aureus is 2 μmol / L.

[0016] The present invention also discloses a pharmaceutical composition, which comprises the above antibacterial polypeptide AuGF or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

[0017] The above pharmaceutical composition can be used in the preparation of drugs for treating and / or preventing bacterial infections.

[0018] The present invention also discloses an antibacterial additive, which comprises the above-mentioned antibacterial polypeptide or its medicinal salt; it can be widely applied to daily necessities, such as disinfectants, detergents, skin care products, etc., to achieve antibacterial and antiseptic effects.

[0019] Advantages: Compared with the prior art, the present invention has the following remarkable advantages:

[0020] (1) The antibacterial polypeptide AuGF of the present invention has broad-spectrum antibacterial activity and has significant antibacterial effects on various bacteria, including Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, and methicillin-resistant Staphylococcus aureus, etc.; moreover, it improves the antibacterial efficiency against drug-resistant bacteria and broad-spectrum pathogens, can play a significant role at low concentrations, has a low minimum inhibitory concentration against various pathogenic bacteria, and has strong antibacterial ability. Among them, the minimum inhibitory concentration against Staphylococcus aureus is only 1 μmol / L, which is beneficial to reducing side effects and improving the safety of drugs;

[0021] (2) The antibacterial polypeptide AuGF of the present invention can effectively address the problem of bacterial drug resistance. It has excellent inhibitory effects on clinical drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus and has good clinical application prospects, while antibiotics at the same concentration do not have anti-methicillin-resistant Staphylococcus aureus activity;

[0022] (3) The antibacterial polypeptide AuGF of the present invention and its pharmaceutical compositions can not only be used for the treatment and prevention of bacterial infections, but also be used as antibacterial additives in the medical and daily chemical fields, providing effective antibacterial means for the healing of infectious wounds, skin care, and daily cleaning, and having broad market application prospects. Its advantages are as follows:

[0023] High efficiency: It shows better effects than traditional antibiotics (cefoxitin and norfloxacin) in antibacterial experiments;

[0024] Stability: It can still maintain stable activity in complex environments (such as neutral pH and normal temperature);

[0025] Rapid antibacterial effect and the ability of rapid bactericidal: It is confirmed by scanning electron microscope observation that the antibacterial polypeptide AuGF can kill bacteria (99.9%) within 2 hours after contacting bacteria.

[0026] Reduced production cost (the AuGF sequence is a short-chain antibacterial peptide with a length of 15, and common amino acids are used). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The growth inhibitory effect of different concentrations of AuGF on Staphylococcus aureus;

[0028] Figure 2Growth inhibitory effects of AuGF at different concentrations on Streptococcus mutans and Actinobacillus actinomycetemcomitans;

[0029] Figure 3 Growth inhibitory effects of AuGF at different concentrations, cefoxitin (FOX), and norfloxacin (NFX) on methicillin-resistant Staphylococcus aureus;

[0030] Figure 4 AuGF at a concentration of 5 μmol / L rapidly causes large-scale rupture of the cell membrane of Staphylococcus aureus. Detailed implementation manners

[0031] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The test materials used in the embodiments can be obtained through conventional channels.

[0032] Strains used: The Staphylococcus aureus ATCC29213, Streptococcus mutans ATCC25175, Actinobacillus actinomycetemcomitans ATCC43717, and methicillin-resistant Staphylococcus aureus ATCC43300 required for the experiment are widely used strains in domestic and foreign laboratories and are strains preserved in this laboratory.

[0033] Main reagents: The antibacterial polypeptide AuGF used was synthesized by Nanjing Genscript Biotech Co., Ltd.; the antibiotics cefoxitin and norfloxacin were purchased from Shanghai Macklin Biochemical Co., Ltd.

[0034] Instrumentation: Microplate reader MultiskanTM FC, Thermo Fisher Scientific; pipette, Eppendorf; scanning electron microscope, Carl Zeiss AG, etc.

[0035] Example 1

[0036] The antibacterial polypeptide AuGF of the present invention, named Jinge, has the amino acid sequence SEQ ID NO.1: GLFIKIIKIKIAKSF. The ability of the antibacterial polypeptide AuGF to inhibit biofilm formation was verified by a crystal violet staining experiment. The experimental procedure is as follows:

[0037] (1) Cultivate Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, and methicillin-resistant Staphylococcus aureus in fresh broth (LB) medium (37 °C, shaker at 180 rpm) until the logarithmic growth phase is reached.

[0038] (2) Centrifuge the bacterial solution obtained in (1), collect the bacterial precipitate, and wash it 3 times with physiological saline. Finally, a bacterial solution with a concentration of 10 5 CFU / mL is obtained for later use.

[0039] (3) Staphylococcus aureus was co-cultured with the antimicrobial polypeptide AuGF at different concentrations (0.5, 1, 2 μmol / L) in a cell culture plate for 48 hours. Streptococcus mutans and Actinobacillus actinomycetemcomitans were co-cultured with the antimicrobial polypeptide AuGF at different concentrations (1, 2, 5 μmol / L) in a cell culture plate for 48 hours. Methicillin-resistant Staphylococcus aureus was co-cultured with the antimicrobial polypeptide AuGF at different concentrations (1, 2, 5 μmol / L), cefoxitin, and norfloxacin in a cell culture plate for 48 hours. The amount of bacterial solution added to each sample well was 100 μL, and mature biofilms were formed by the bacteria after 48 hours.

[0040] (4) Discard the culture medium in the sample wells and rinse three times with phosphate buffer solution (PBS).

[0041] (5) Add methanol solution to the sample wells to fix the biofilms for 15 minutes, then aspirate the remaining methanol and air dry.

[0042] (6) Add 0.1% (v / v) crystal violet solution, let it stand for 15 minutes, then rinse with PBS and air dry.

[0043] (7) Add 95% (v / v) ethanol, let it stand at 37 °C for 30 minutes until the attached crystal violet dye is completely dissolved.

[0044] (8) Use a microplate reader to record the OD 595 value.

[0045] (9) Calculate the bacterial survival rate. Bacterial survival rate = (A m - A n ) / (A p - A n ) × 100%

[0046] where A m is the OD value of the experimental group, A p is the OD value of the positive control, and A n is the OD value of the negative control.

[0047] The results are as Figure 1 shown. After co-culturing Staphylococcus aureus with the antimicrobial polypeptide AuGF at a concentration of 0.5 μmol / L for 48 hours, mature biofilms were formed in the sample wells and showed dark blue after staining. After co-culturing Staphylococcus aureus with the antimicrobial polypeptide AuGF at a concentration of 1 μmol / L for 48 hours, no biofilms were formed in the sample wells, indicating that the antimicrobial polypeptide AuGF has significant anti-Staphylococcus aureus activity at the current concentration.

[0048] As Figure 2As shown, after Streptococcus mutans was co-cultured with the antimicrobial polypeptide AuGF at a concentration of 2 μmol / L for 48 hours, a mature biofilm was formed in the sample wells and showed dark blue after staining. After Streptococcus mutans was co-cultured with the antimicrobial polypeptide AuGF at a concentration of 5 μmol / L for 48 hours, no biofilm was formed in the sample wells, indicating that the antimicrobial polypeptide AuGF has significant anti-Streptococcus mutans activity at the current concentration.

[0049] As Figure 2 shown, after Actinobacillus actinomycetemcomitans was co-cultured with the antimicrobial polypeptide AuGF at a concentration of 2 μmol / L for 48 hours, a mature biofilm was formed in the sample wells and showed dark blue after staining. After Actinobacillus actinomycetemcomitans was co-cultured with the antimicrobial polypeptide AuGF at a concentration of 5 μmol / L for 48 hours, no biofilm was formed in the sample wells, indicating that the antimicrobial polypeptide AuGF has significant anti-Actinobacillus actinomycetemcomitans activity at the current concentration.

[0050] As Figure 3 shown, after methicillin-resistant Staphylococcus aureus was co-cultured with the antimicrobial polypeptide AuGF at a concentration of 1 μmol / L for 48 hours, a mature biofilm was formed in the sample wells and showed dark blue after staining. After methicillin-resistant Staphylococcus aureus was co-cultured with the antimicrobial polypeptide AuGF at a concentration of 2 μmol / L for 48 hours, no biofilm was formed in the sample wells, indicating that the antimicrobial polypeptide AuGF has significant anti-methicillin-resistant Staphylococcus aureus activity at the current concentration.

[0051] As Figure 3 shown, after methicillin-resistant Staphylococcus aureus was co-cultured with the antibiotic cefoxitin at a concentration of 5 μmol / L for 48 hours, a mature biofilm was formed in the sample wells and showed dark blue after staining. After methicillin-resistant Staphylococcus aureus was co-cultured with the antibiotic norfloxacin at a concentration of 5 μmol / L for 48 hours, a mature biofilm was formed in the sample wells and showed dark blue after staining. The results indicate that the antibiotics cefoxitin and norfloxacin do not have anti-methicillin-resistant Staphylococcus aureus activity at the current concentration.

[0052] Among the above bacteria, Staphylococcus aureus, Streptococcus mutans, and methicillin-resistant Staphylococcus aureus are Gram-positive bacteria, and Actinobacillus actinomycetemcomitans is a Gram-negative bacteria. Methicillin-resistant Staphylococcus aureus is a drug-resistant bacterium. The above experimental results show that the antimicrobial polypeptide AuGF can effectively inhibit the growth of selected Gram-negative and Gram-positive bacteria. Compared with the selected antibiotics, the antimicrobial polypeptide AuGF can effectively inhibit the growth of selected drug-resistant bacteria.

[0053] Example 2

[0054] The killing ability of the antimicrobial polypeptide AuGF against bacteria was verified by taking scanning electron microscope images. The experimental operations are as follows:

[0055] (1) Culture Staphylococcus aureus in fresh broth (LB) medium (37 °C, shaker at 180 rpm) until it reaches the logarithmic growth phase.

[0056] (2) Centrifuge the bacterial solution obtained in (1), collect the bacterial precipitate, and wash it 3 times with physiological saline. Finally, obtain a bacterial solution with a concentration of 105 CFU / mL for later use.

[0057] (3) Co-culture Staphylococcus aureus and the antibacterial polypeptide AuGF with a concentration of 5 μmol / L in a cell culture plate for 2 hours.

[0058] (4) After the bacterial samples are fixed with 4% glutaraldehyde at 4 °C overnight, dehydrate them through an ethanol gradient, drop them on a silicon wafer, and observe their morphology using a scanning electron microscope.

[0059] As Figure 4 shown, after 2 hours of incubation, the surface of the bacteria in the untreated group (Control) is smooth and spherical, indicating that the bacteria are in good growth condition. In the AuGF experimental group, no intact bacteria are observed at all, and the entire area is filled with leaked contents. Figure 4 The white scale bar in

[0060] Therefore, the antibacterial polypeptide AuGF of the present invention has broad-spectrum antibacterial activity. It has significant antibacterial effects against a variety of bacteria, including Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, and methicillin-resistant Staphylococcus aureus, etc., and can effectively address the problem of bacterial drug resistance, having good clinical application prospects.

Claims

1. An antibacterial polypeptide AuGF, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO.1: GLFIKIIKIKIAKSF.

2. Use of the antibacterial polypeptide AuGF or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a medicament for treating and / or preventing bacterial infections.

3. The application according to claim 2, wherein The bacteria are Gram-positive bacteria and / or Gram-negative bacteria.

4. The application according to claim 2, wherein The bacteria include one or more of Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans or methicillin-resistant Staphylococcus aureus.

5. The application according to claim 4, characterized in that The minimum inhibitory concentration of the antibacterial polypeptide AuGF against Staphylococcus aureus is 1 μmol / L.

6. The application according to claim 4, characterized in that The minimum inhibitory concentration of the antibacterial polypeptide AuGF against Streptococcus mutans or Actinobacillus actinomycetemcomitans is 5 μmol / L.

7. The application according to claim 4, wherein The minimum inhibitory concentration of the antibacterial polypeptide AuGF against methicillin-resistant Staphylococcus aureus is 2 μmol / L.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibacterial polypeptide AuGF or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable carrier or excipient.

9. Use of the pharmaceutical composition as claimed in claim 8 in the preparation of a medicament for treating and / or preventing bacterial infections.

10. An antibacterial additive, characterized in that, The antibacterial additive comprises the antibacterial polypeptide or a pharmaceutically acceptable salt thereof as claimed in claim 1.

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